Etiologic Risk Group (see link below): Risk Group 2 Parasitic Agent
*Background/Disease Information (sort of like the Intro to your Mini Research Write up):
Toxoplasmosis is a disease found throughout the word that is caused by a single-celled , obligate intracellular, parasitic parasitic protozoan, T. gondii. The way people get the disease is from ingestion of any under-cooked, contaminated meat, and not washing hands thoroughly after handling it. Symptoms of toxoplasmosis including swollen lymph glands that feel like the flu, or long lasting pains and muscle aches.
Essentiality of this protein:
"This entry represents dual specificity protein-tyrosine phosphatases. Ser/Thr and Tyr dual specificity phosphatases are a group of enzymes with both Ser/Thr (EC:3.1.3.16) and tyrosine specific protein phosphatase (EC:3.1.3.48) activity able to remove both the serine/threonine or tyrosine-bound phosphate group from a wide range of phosphoproteins, including a number of enzymes which have been phosphorylated under the action of a kinase. Dual specificity protein phosphatases (DSPs) regulate mitogenic signal transduction and control the cell cycle. The crystal structure of a human DSP, vaccinia H1-related phosphatase (or VHR), has been determined at 2.1 angstrom resolution [PMID: 8650541]. A shallow active site pocket in VHR allows for the hydrolysis of phosphorylated serine, threonine, or tyrosine protein residues, whereas the deeper active site of protein tyrosine phosphatases (PTPs) restricts substrate specificity to only phosphotyrosine. Positively charged crevices near the active site may explain the enzyme's preference for substrates with two phosphorylated residues. The VHR structure defines a conserved structural scaffold for both DSPs and PTPs. A "recognition region" connecting helix alpha1 to strand beta1, may determine differences in substrate specificity between VHR, the PTPs, and other DSPs."
Source: Dual specificity phosphatase, catalytic domain (IPR000340) (http://www.ebi.ac.uk/interpro/entry/IPR000340)
Complex of proteins?: secrete phosphotyrosine phosphatase
--- List cost and quantity of substrate reagents, supplier, and catalog #
Substrate reagent: sulfadoxin [S7821]
10G: $68.50
25G: 169.00
Supplier: Sigma-Aldrich
CAS Number: 2447-57-6
Structure Available (PDB or Homology model)
-- PDB # or closest PDB entry if using homology model: 2ISN (not very similar actually - Dr. B 091114)
-- For Homology Model option:
---- Show pairwise alignment of your BLASTP search in NCBI against the PDB
---- Query Coverage:
---- Max % Identities:
---- % Positives
---- Chain used for homology:
Inhibitory phosphorylation on Thr and Tyr located in the glycine-rich ATP anchor motif of cdks results in inactivation
Source: Small molecule inhibitors of dual specificity protein phosphatases (http://www.nature.com/onc/journal/v19/n56/full/1204084a.html)
Expression Information (has it been expressed in bacterial cells):
"The conditional expression system to generate a transgenic parasite line in which the levels of T. gondii AMA1 (TgAMA1) expression can be experimentally manipulated."
Source: Conditional Expression of Toxoplasma gondii Apical Membrane Antigen-1 (TgAMA1) Demonstrates That TgAMA1 Plays a Critical Role in Host Cell Invasion ( http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1196342/)
Purification Method:
"Four methods to separate and purify Toxoplasma gondii tachyzoites from in vivo and in vitro culture systems, including trypsin digestion, purification with a 3-μm filter, CF-11 cellulose purification, and Percoll purification."
Source: Separation and purification of Toxoplasma gondii tachyzoites from in vitro and in vivo culture systems (http://www.ncbi.nlm.nih.gov/pubmed/22033076)
Image of protein (PyMol with features delineated and shown separately):
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
">2ISN:A|PDBID|CHAIN|SEQUENCE
SLKKVITVNEWYTTTVAATMLGRRPTDEDAILVSAPATSRPNVRIKAVFDGHAGEATSQYCAKHAAKHLGKLSEFTFAEV
KKACLSLDAEIIRKLGPKHVAGSTGIIVAIERLSAPVVENVVGREIVPRAHEETFVPLEKLIQEEEEAEHPELVGRYPRV
PDVQQKTIPAGSFLVTAINIGDSRATLIHSDGGLTRLSKDHKPNHPTEASRIEKAGGSVETFDVPRVDGVLALSRAFGDS
DFKMNPNLPPEEQKVIAVPDVRQFYALSSDLLLLACDGVYEPSGMDWAYVRDLTVAEMQRSKGDLEEVAARVMDYAYDMN
SQDNISVMLVAFHNQEVEHPTAVYKVVSGQVDKVEWDVAGKKGN
*length of your protein in Amino Acids: 364
Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website:
39850.2 g/mol
Molar Extinction coefficient of your protein at 280 nm wavelength:
Extinction coefficients are in units of M-1 cm-1, at 280 nm measured in water.
Ext. coefficient: 30035 M-1 cm-1
Abs 0.1% (=1 g/l) 0.754, assuming all pairs of Cys residues form cystines
Ext. coefficient: 29910 M-1 cm-1
Abs 0.1% (=1 g/l) 0.751, assuming all Cys residues are reduced
*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):
*GC% Content for gene (codon optimized):
Do Not Need this info for Spring (but still copy these lines to your Target page for now) Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers): (link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):
**
*NCBI Gene # or RefSeq#: 7895273
*Protein ID (NP or XP #) or Wolbachia#: EPT31998.1
*Organism (including strain): Toxoplasma gondii (ME49 )
Etiologic Risk Group (see link below): Risk Group 2 Parasitic Agent
*Background/Disease Information (sort of like the Intro to your Mini Research Write up):
Toxoplasmosis is a disease found throughout the word that is caused by a single-celled , obligate intracellular, parasitic parasitic protozoan, T. gondii. The way people get the disease is from ingestion of any under-cooked, contaminated meat, and not washing hands thoroughly after handling it. Symptoms of toxoplasmosis including swollen lymph glands that feel like the flu, or long lasting pains and muscle aches.
Link to TDR Targets page (if present): http://tdrtargets.org/targets/view?gene_id=259586
Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.)
http://www.ncbi.nlm.nih.gov/gene/?term=TGME49_021590
Essentiality of this protein:
"This entry represents dual specificity protein-tyrosine phosphatases. Ser/Thr and Tyr dual specificity phosphatases are a group of enzymes with both Ser/Thr (EC:3.1.3.16) and tyrosine specific protein phosphatase (EC:3.1.3.48) activity able to remove both the serine/threonine or tyrosine-bound phosphate group from a wide range of phosphoproteins, including a number of enzymes which have been phosphorylated under the action of a kinase. Dual specificity protein phosphatases (DSPs) regulate mitogenic signal transduction and control the cell cycle. The crystal structure of a human DSP, vaccinia H1-related phosphatase (or VHR), has been determined at 2.1 angstrom resolution [PMID: 8650541]. A shallow active site pocket in VHR allows for the hydrolysis of phosphorylated serine, threonine, or tyrosine protein residues, whereas the deeper active site of protein tyrosine phosphatases (PTPs) restricts substrate specificity to only phosphotyrosine. Positively charged crevices near the active site may explain the enzyme's preference for substrates with two phosphorylated residues. The VHR structure defines a conserved structural scaffold for both DSPs and PTPs. A "recognition region" connecting helix alpha1 to strand beta1, may determine differences in substrate specificity between VHR, the PTPs, and other DSPs."
Source: Dual specificity phosphatase, catalytic domain (IPR000340) (http://www.ebi.ac.uk/interpro/entry/IPR000340)
Complex of proteins?: secrete phosphotyrosine phosphatase
Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism):
Cdc25
Source: Protein tyrosine phosphatases: mechanisms of catalysis and regulation (http://www.sciencedirect.com/science/article/pii/S1367593198800951)
*EC#: 3.1.3.48
Link to BRENDA EC# page:
http://www.brenda-enzymes.org/php/result_flat.php4?ecno=3.1.3.48
-- Show screenshot of BRENDA enzyme mechanism schematic
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents):
-- link to Sigma (or other company) page for assay (see Sigma links below)
http://www.sigmaaldrich.com/catalog/search?interface=All&term=toxoplasma%20gondii%20phosphatase&N=0&focus=product&lang=en®ion=US
-- -or link (or citation) to paper that contains assay information
http://www.sigmaaldrich.com/catalog/papers/10779791
-- links to assay reagents (substrates) pages.
http://www.sigmaaldrich.com/catalog/product/sigma/s7821?lang=en®ion=US
--- List cost and quantity of substrate reagents, supplier, and catalog #
Substrate reagent: sulfadoxin [S7821]
10G: $68.50
25G: 169.00
Supplier: Sigma-Aldrich
CAS Number: 2447-57-6
Structure Available (PDB or Homology model)
-- PDB # or closest PDB entry if using homology model: 2ISN (not very similar actually - Dr. B 091114)
-- For Homology Model option:
---- Show pairwise alignment of your BLASTP search in NCBI against the PDB
---- Query Coverage:
---- Max % Identities:
---- % Positives
---- Chain used for homology:
Current Inhibitors:
two heat-stable proteins known as Inhibitor-1 (I-1) and Inhibitor-2 (I-2)
Source: Protein Phosphatase Inhibitors ( http://www.emdmillipore.com/life-science-research/protein-phosphatase-inhibitors/c_Sc2b.s1L.woAAAEWyGEfVhTm)
Inhibitory phosphorylation on Thr and Tyr located in the glycine-rich ATP anchor motif of cdks results in inactivation
Source: Small molecule inhibitors of dual specificity protein phosphatases (http://www.nature.com/onc/journal/v19/n56/full/1204084a.html)
Expression Information (has it been expressed in bacterial cells):
"The conditional expression system to generate a transgenic parasite line in which the levels of T. gondii AMA1 (TgAMA1) expression can be experimentally manipulated."
Source: Conditional Expression of Toxoplasma gondii Apical Membrane Antigen-1 (TgAMA1) Demonstrates That TgAMA1 Plays a Critical Role in Host Cell Invasion ( http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1196342/)
Purification Method:
"Four methods to separate and purify Toxoplasma gondii tachyzoites from in vivo and in vitro culture systems, including trypsin digestion, purification with a 3-μm filter, CF-11 cellulose purification, and Percoll purification."
Source: Separation and purification of Toxoplasma gondii tachyzoites from in vitro and in vivo culture systems (http://www.ncbi.nlm.nih.gov/pubmed/22033076)
Image of protein (PyMol with features delineated and shown separately):
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
">2ISN:A|PDBID|CHAIN|SEQUENCE
SLKKVITVNEWYTTTVAATMLGRRPTDEDAILVSAPATSRPNVRIKAVFDGHAGEATSQYCAKHAAKHLGKLSEFTFAEV
KKACLSLDAEIIRKLGPKHVAGSTGIIVAIERLSAPVVENVVGREIVPRAHEETFVPLEKLIQEEEEAEHPELVGRYPRV
PDVQQKTIPAGSFLVTAINIGDSRATLIHSDGGLTRLSKDHKPNHPTEASRIEKAGGSVETFDVPRVDGVLALSRAFGDS
DFKMNPNLPPEEQKVIAVPDVRQFYALSSDLLLLACDGVYEPSGMDWAYVRDLTVAEMQRSKGDLEEVAARVMDYAYDMN
SQDNISVMLVAFHNQEVEHPTAVYKVVSGQVDKVEWDVAGKKGN
*length of your protein in Amino Acids: 364
Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website:
39850.2 g/mol
Molar Extinction coefficient of your protein at 280 nm wavelength:
Extinction coefficients are in units of M-1 cm-1, at 280 nm measured in water.
Ext. coefficient: 30035 M-1 cm-1
Abs 0.1% (=1 g/l) 0.754, assuming all pairs of Cys residues form cystines
Ext. coefficient: 29910 M-1 cm-1
Abs 0.1% (=1 g/l) 0.751, assuming all Cys residues are reduced
TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.
*CDS Gene Sequence (paste as text only):
>gi|212659404:449825-453956 Toxoplasma gondii ME49 scf_1112359875116, whole genome shotgun sequence
GAAAGGAAGACGGCAACATATTCGCGACGAATTCGGTGCATGCCTTCTAACTGGTTGTGTCTCTCCTCCC
GTTCGTTAATACACTATGCTTGCCTCGGCCGAATGTTCTCAATCCCCCGCGGCCGCAGAACTGTTTCAAA
ATAGAGAGACGAGACTAGAGCTGCGCGTCTCTTTTTGAGGCCGACCCGGTGTCATTTTTTTACAAGGGGA
TTATGAAGTCGTGATTACAGCCTTTTTTTGACAAAGAGCTAAGGTCGCTGCTATGGTAATAAGAACGAAA
ACCGTGTAAAGTGACCGGCGTTTCATCCTCCCTCTTGCGCTGACACAAAGTGACGCGGTGATCCAGCTTG
ATTGAGTGACCTCTTCTTATTGTGCCTCTCTTTCAATTCATGCAAAAGCGTTCCCACGTCTGTACAGCCG
TATCTGCATTCTGTTTTTGTGTCCCAGCCTGCTTCAAGCCAGTCAGTTGTGTAACGGTAAATGGAGGTGG
CCGTTTCTTCCTCGGGACCTTCTTCCCTCCGCCCTGTCTCGGTCTACCACGGTGGAGCAAGCGGGTCTGA
GGATAAGAAACTGTGGATCTGGCTACCGTACATTTCTGCATTATCGCGCAACGGCTTCGGCACCTCGAAT
AATTCGCAGTTGGCATCCCGCGGCTCTCTGCGCCCCTCCGTGGCGTCAATGCAGAGTGCTTCTTCTGCCG
TTGGAGGTTCACTACCAGGAGTATCTTCGTCGGTGAACACATCCGCGGATGAGACGTGGGAAACAGTACG
CAATATGGTTGTTGGAAGGCCAGTGACTCTTCAGTCCGCGGAGCGAATCCGAGCAGGGTCAGCACCCGCT
AGCAGTCTCATGCCGAAGTGTCCCACAGACATCTATCCTCCGGGTTTGGTACCCACTCTGACGAAAGGTA
GCCTTTTTGTGGGGTCCCTGAAGCACGCACTCGACGAGAACCTTCTCCTAAAATTCAACGTGAAACTCGT
TGTTACAGTTGCGTGGCCATACGGAAGTTGGCCGCTTCAACAGCGCGTGCTCTATTCTCGTCTTGGGATC
AGCCACATCAACCACCCTCTCTTGGATTCTCCGTCCCAGGTGAGCTCAGCACACAGAAGTCGGTAACGTT
GTGACCGTGGCGGACGGAGGCACACGCGACAGGGTGGAAACGGTGCGTTTGGTTAGACTTCACTTTTAGG
AGGTAAAACCGCTTGACGTGGGCTCGCTAGCGGCTCGAGACAAATTTTGCACATGGACTCATGCAAACGG
ACTTCCCTGTGAAATGAGACAGTGAACCGAGGATTGTAATGCGTTCTTTCAAGCCCAACTTGCTCGTGAA
TAACGTCTTCAGTGTTAGTCTGGCAAGGATGTTGTACCGAATGAGATTGTGAACTCACGCTTGCTGGGAT
TGGGTTCGCTGCGTTTTTGTTAGGAGCTCGACTTTTCCCGCCTCTCATTGGCACGTATTCACTCATACCT
CGCCAGGTAAGTGGGCATCGACTGTATACCGCAATTCCTTGTTGGGTTTTCTTCTGTTCATTCATGGCCC
AAATTTCAGGTTTCCGTCACGAAGTTGATAGGTTTTTCCCCTCATCAGAGCAGATGTGTACCTCCGGTTT
CACCTGCAGGAACTACGATAAGCAGTTGCTTACTGGTGGACACAGGGAAATTGAATATGGACTCTGCCAG
TGATGGTGTTAATCCTTTTCTATTAATCGTTCCCGCGGTCAATTTATAACATTCCCCGCATACGTTGTAG
CACTTCTGCGTAAGACAGAACCACTACTGGCTCGTCTTCCGTGTCATGCCCGTGAAATCGTCGTTGTACT
GTCGCATAGGAGTTGCTTTTTCTTTATCGAGTGACTAGACGCGATTCATCTGTGAGGCGTGGCATTTTAG
CGAGTGATGTTGCACTTTTACACACATGCATGCAACTCACCCTTTTTAACCAAATGTGTGTATATGCGCG
GGCACACGCATATGATGGCATTAGTACGTTGTGTGTATTAAAACGGGTTGTGAGTTATTGACGCGGCCCC
CAACTGGGATGTGAAGGTGAAATTTTCGAGTGCGGCAAGCATTTTGAATGTGTTGCTCTTGTTTTTGCGC
CGACTGCATCTCTTGCAGGGGAGTGACAGTGTTAGTTCACTGTGAAAAGGGCATCTCTCGGTCTGTCTCC
CTCTGTGCGGTGAGCGAAGATCTGTTACAGCTTCCAAATCAACACCGTTCGAAGCCTTCTGCTCTTCTTA
TGTGTAATAACTATCTACGTTAAGCTTTTTGCACTGCGAATAACTTGTATGCCTTGCCTGAAGAATACAC
TTGTTAAGGCAGAAAAGCAGGTAATCTGGTCAGCAGTGGCACATGACGACAGCATATGCTGGACTCGGGT
TCTTCCTTTCAATTGGAGAGGCACAGATGTGAGAACATACTGGCACATTGATGTGATTCGAGTACGTGTG
GCTTTGTAGTGTCCTCAGAGTCCAGTTTGATCGAATAGGCATTCAAAACCTCGGAACACAGCTGGTGGAC
TTCCAATGTGACAACGCGTTTGAATAATTTCTTTTCGACGTATGGCCTGTTGTATGGCAACAAATGGATT
TCATGCGTGCGATTTTCATGGCAGGCATATCTCATCGTTTATCATGGGCACACAACCATGTCTGCGTTGG
AGGCAATTCGTAAATATCGCCCCATTGCGCGGCCGAATCCCGGGTTTTTTGTGCAACTCCAACGACTAGA
AAGCAATCATCACACGGTGACAGCGTCTTTTCAAAACAGCTCTAACTCGCCATCAAGGCGCTCCCCTTCG
AGAGTCGGTACGGAATTATCTAGTGGACGCTGATTCGGGATCTGTGGTGGGAAAAGCACATATCCCTATG
GCAGGAATAGAGGTCGTCTAACGGTTATCAGTGTATACACCTTTCGTACTTATGTGTTTGCTTTGTGTCT
CTTCTTCATTTGCCCATAGACTGGCCCCACTTCCACTGCTTCCCCACCTTCTCCTTCCCCTGTCGTCCTG
AAGGGTCCTGTGTTGGGTCCAAGGAGGAGCGTATAATGTTGATGCGTCAGGGAACAAAAGAGAAGGTGTT
GCAGAGCGGTAAACGGGCAGAGATGGAGTTGTGAACGGTGACGTATGAGGGTCCAGCATCGATACACTCA
ATGAGAGACTGGCACTGAGCACGGCATGGAACTGCCGCTTCAGACTATGCAATTATTTGCTGGTCACCAG
TCGAAGCACTTGCTGGCCTCTGGTGAGTCTCCGGTGGATGCGATCGGCAGACGAATGCGAAGAAGACGGT
GCTTCAAGCACGCGACATCCCGCGCAAGATGGTCCAGACACCGTGTGCTTCAGTGGCAATTCAGGTTTAA
AAAGTACCGATCTTCAGATGTATAGCGCTGCACCGGAGTAATCCATGTATCGACGTCGTGCGACACTGCT
AGCAGGAAAGGCAGTCGTATTCGCCAGCAAGTGCATCTCGTAACTCGTTGTGAACAGGTGGTTAAGTGTG
ACGTCCATGAGAAATTGACACCATTTTTGCATTCTCGCCTTAGTTTTAGGTCGCAACTTCGATTCTATAT
GCAAGAATTTTCCCATAGCAACGGTCACAGTGTGCCCGTCGAGTTCTGAGTGTCTTAACCCCGACGAGTT
TAGTGTGGTACACGGGGCCGCCATGTCGGGGTATAGACCTACGGATCCTTAGCTGAAAAAATAGAAAAGA
CGACGGTGTGCGTGTGTGTTGGCAGTTGAGCAGGTGGTTGTCGTTGCATATGCGCTGACTTATGCGCCGA
ATGGTCAATGCGATGGGCCGGTGTCACTCGTTAATCCGTCTTTTGTGAAGCGGTCCAGAATTAAGCAGAA
CTTTGTGTAAAGGATTTTGATAGTCGTCACCGCCGCCTATACACTGTAGGTATTTTTGTCCAGGCTTGTC
GGTTCCGTAGGTTTCCCAATGAGGTGTTTCTAGCGTGAGAACTGCGTTCCAGTCGTGTTGCTTCACTTGG
ATATGCCCCCCAACCACTGCCCGAGCAAAGTGCTGCTTGTCACGTGCGGTGGAAGCGTTTTTTCTCGAAA
GGTAGATTGTCGTCGTTGTAAGGACCTGTTTTCGAGATTTTAGTGTTACGGGTTCAATTTTAGCGACTTT
CG
*GC% Content for gene: 7.14%
*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):
*GC% Content for gene (codon optimized):
Do Not Need this info for Spring (but still copy these lines to your Target page for now)
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):
(link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):
**
Resources:
See ProtocolTargetDiscoveryVDS.docx for moreEtiologic Risk Group Categories (for pathogens): http://www.utexas.edu/research/rsc/ibc/agent_class.html#_Toc7238334
SIGMA-ALDRICH RESOURCES
Enzyme Explorer
http://www.sigmaaldrich.com/life-science/metabolomics/enzyme-explorer.html
Enzyme Classification Index (EC number)
http://www.sigmaaldrich.com/life-science/biochemicals/biochemical-products.html?TablePage=14573088
WolframAlpha http://www.wolframalpha.com/
DrugBank http://www.drugbank.ca/
Databases of genes/organisms:
http://www.niaid.nih.gov/Pages/default.aspx
http://eupathdb.org/eupathdb/
https://patricbrc.vbi.vt.edu/portal/portal/patric/Home
http://www.nmpdr.org/FIG/wiki/view.cgi/Main/EssentialGenes
http://tubic.tju.edu.cn/deg/
http://csgid.org/csgid/cake/pages/community_request_gateway
http://tdrtargets.org/
http://gsc.jcvi.org/status.shtml
Scientific Nomenclature page from Center for Disease Control (gene, protein names and abbreviations)
http://wwwnc.cdc.gov/eid/pages/scientific-nomenclature.htm
Gene Information:
NCBI GENE Page: http://www.ncbi.nlm.nih.gov/gene
BLAST Page: http://blast.ncbi.nlm.nih.gov/
Protein Information:
NCBI Protein Page: http://www.ncbi.nlm.nih.gov/protein
Protein Expression Website
Protein Expression Paper: SGC_ProteinProductionPurificationNatMethods2008.pdf
Primer Overlap PCR Articles
HooverLubkowski_PCRoverlapcloninggnf042.pdf
StemmerPCRoverlapGene1995.pdf
Is my target good for Virtual Screening programs?
Reynolds_THermodynamicsLigandBinding_MedChemLett2011.pdf